A considerable amount of attention has been directed towards the use of monoclonal antibody-enzyme conjugates in combination with suitable prodrugs for the selective delivery of chemotherapeutic agents to tumors (reviewed in Senter et al., Bioconiugate Chem., 4:3-9 (1993); Jungheim et al., Chem Rev., 94:1553-1566 (1994); Bagshawe et al., Ann. Oncol., 5:879-891 (1994)). The monoclonal antibody (mAb) portions of these immunoconjugates recognize tumor-selective antigens and are capable of delivering the enzymes to tumor masses. Once tumor localization and systemic conjugate clearance has taken place, a non-cytotoxic prodrug form of a chemotherapeutic drug is administered which is converted into an active drug by the targeted enzyme. This leads to the selective delivery of anticancer drugs to sites of neoplasia. Pharmacokinetic studies have shown that the intratumoral drug concentrations resulting from mAb-enzyme/prodrug combinations can be significantly greater than that achieved by systemic drug administration (Bosslet et al., Cancer Res., 54:2151-2159 (1994); Svensson et al., Cancer Res., 55:2357-2365 (1995); Wallace et al., Cancer Res., 54:2719-2327 (1994)). This probably accounts for the observed antitumor activities, which include complete tumor regressions and cures in a number of different models for human cancer (Springer et al., Eur. J. Cancer, 27:1361-1366 (1991); Meyer et al., Cancer Res., 53:3956-3963 (1993); Eccles et al., Cancer Res., 54:5171-5177 (1994); Kerr et al., Cancer Res., 55:3558-3563 (1995)).
Kerr et al. disclosed the use of antibodies against the human p97 (melanotransferrin) tumor antigen for the delivery of .beta.-lactamase (bL) to tumor cells (Kerr et al., Cancer Res., 55:3558-3563 (1995)). This antigen has been found to be overexpressed on a majority of clinical melanoma isolates and is also observed on many human carcinomas (Woodbury et al., Proc. Natl. Acad. Sci. (USA), 77:2183-2187 (1980); Brown et al., J. Immun., 127:539-545 (1981), Brown et al., Proc. Natl. Acad. Sci. (USA), 78:539-543 (1981); Rose et al., Proc. Natl. Acad. Sci. (USA) (1986)). Significant antitumor activities have been obtained using the combination of a chemically produced anti p97-Fab'-bL conjugate in combination with CCM (7-(4-carboxybutan-amido)cephalosporin mustard; Kerr et al., Cancer Res., 55:3558-3563 (1995)), a cephalosporin containing prodrug of phenylenediamine mustard (PDM). These effects were observed in a melanoma tumor model that was resistant to the activities of PDM.
A major concern in the development of mAb-bL conjugates for clinical testing is conjugate uniformity. Typically, mAb-enzyme conjugates are prepared using bifunctional cross-linking reagents that react in a random fashion with exposed amino acid residues on the individual proteins. Immunoconjugates produced in this manner are microscopically heterogeneous due the inherent lack of specificity of the cross-linking reagents. In addition, chemically prepared conjugates are typically isolated in low yields. Although recent reports describe alternative coupling chemistries that can afford higher yields of more homogeneous immunoconjugates (Mikolajczyk et al., Bioconjugate Chem., 5:636-646 (1994); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Werlen et al., Tumor Targeting, 1:251-258 (1995)), these methods still involve chemical modification steps that can contribute to product heterogeneity.
Genetically constructed fusion proteins offer an alternative method of producing homogeneous mAb-enzyme conjugates. There have now been reports describing the production, characterization, and activities of recombinant Fab, sFv, and disulfide stabilized Fv-enzyme fusion proteins (Bosslet et al., Br. J. Cancer, 65:234-238 (1992); Goshorn et al., Cancer Res., 53:2123-2127 (1993); Rodrigues et al., Cancer Res., 55:63-70 (1995)).
Thus, a need exists for homogeneous mAb-enzyme conjugates for use in combination with prodrugs. The instant invention addresses this need and more.